Accumulating evidence shows that many neurons release two classical neurotransmitters, but fundamental questions remain about the cellular basis for corelease, with important implications for its physiological role. In this proposal, we use the vesicular neurotransmitter transporters to elucidate the mechanisms involved in corelease. In previous work, we showed that glutamate corelease by midbrain dopamine neurons serves two distinct roles, one in vesicle filling with dopamine and the other as an independent signal. Although the effects on vesicle filling require colocalization of the vesicular monoamine transporter VMAT2 and vesicular glutamate transporter VGLUT2 on the same synaptic vesicles, anatomy has suggested some segregation as well, but with unclear physiological consequences. We now find that dopamine neurons release glutamate and dopamine with different properties. Release of the two transmitters differs in short-term depression and depends on different presynaptic Ca++ channels. Synaptic vesicles belong to pools that differ in response to stimulation but these differences have been attributed to extrinsic factors such as cytoskeletal association. We now show that they also differ in composition because they contain different transmitters and release them with different properties. Through this mechanism, a single neuron can deconvolve its input into two distinct outputs. The long-term objectives of this project are to elucidate the cellular and molecular basis for neurotransmitter corelease and determine its role in information processing. The strategy is to use the vesicular transporters to characterize the different vesicle populations. Specifically, we will 1) compare monoamine and glutamate release by imaging VMAT2 and VGLUT2 in live neurons; 2) determine how VMAT2 and VGLUT2 target to distinct vesicle populations; 3) characterize the composition of monoamine and glutamate SVs by proteomics. The results will provide basic information about the organization of neurons, with direct relevance for corelease by other cells, but also for release by all neurons.